Tuberculosis remains a major global public health crisis despite being a curable disease. One characteristic feature of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is its slow growth. Mtb employs multiple regulatory networks for maintaining its optimal growth in vivo and one such network is the MtrAB two-component regulatory signal transduction system (2CRS). The consequences of Mtb-host interactions dictate whether it maintains an active replicative state with increase in bacterial burden, or remains metabolically active, but in a non-replicative persistent (NRP) state. DNA replication is essential for the multiplication of organisms and this process is believed to be regulated during intracellular growth. The genetic elements responsible for regulation of Mtb replication and or the factors that promote NRP state in vivo are largely unknown. Our proposal focuses on DNA replication. DnaA protein initiates this process at a unique site on the genome called oriC. MtrA response regulator (RR) binds to oriC and dnaA promoter suggesting that DnaA mediated oriC is subject to regulation by the MtrAB 2CRS. DnaA associates with membranes and the ability of DnaA to initiate replication depends, in part, on its interactions with membrane lipids, possibly modulated by LysX, a hitherto unrecognized virulence factor. We propose that in response to infection two divergently different pathways[unreadable]one mediated by the MtrAB 2CRS and the other by the LysX, regulate DnaA activity, hence replication initiation. A consequence of this regulation determines whether DNA replication will continue resulting in active multiplication, or stall/shut-down contributing to NRP state. We will dissect both pathways in an attempt to define their roles on DnaA activity and replication initiation during intracellular growth. Specific Aim 1 focuses on the roles of the MtrAB signal transduction pathway on DnaA mediated oriC replication during intracellular growth using biochemical and genetic approaches. Proposed experiments will evaluate MtrA occupancy of oriC under synchronous replication conditions, and evaluate how DnaA mediated oriC replication is regulated by the MtrAB system upon infection. Specific Aim 2 examines if lysX is a unrecognized virulence factor, and if it regulates the production of lysinylated polar lipids, thereby influences membrane fluidity, hence DnaA activity. A long term goal of these studies is to understand how the LysX regulated changes in membrane fluidity result in replication arrest and induction of Mtb persistence. It is hoped that these experiments will define how the DNA replication process in Mtb is regulated, hence will improve our understanding of proliferation of Mtb in vivo.